Of the genes known to be involved in human cancer, mutations have been most commonly detected in the tumor suppressor gene p53. The p53 gene product activates transcription of many downstream genes, and by regulating their transcription, exerts a variety of biological functions. The two main functions of p53 are cell cycle arrest and apoptosis, mediated by p21Waf1 and BAX respectively. These functions were thought to be characteristics essential for p53-dependent tumor suppression (el-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Parsons, R., Trent, J. M., Lin, D., Mercer, W. E., Kinzler, K. W., and Vogelstein, B. (1993). WAF1, a potential mediator of p53 tumor suppression. Cell 75, 817-825; Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K., and Elledge, S. J. (1993). The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75, 805-816; Miyashita, T. and Reed, J. C. (1995). Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 80, 293-299).
The present inventors recently discovered p53R2, a novel p53 target molecule that supplies deoxyribonucleotides for DNA repair. Thus, p53 is also directly involved in DNA repair (Tanaka, H., Arakawa, H., Yamaguchi, T., Shiraishi, K., Fukuda, S., Matsui, K., Takei, Y., and Nakamura, Y. (2000). A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage. Nature 404, 42-49).
To date, it has been reported that a large number of p53 target genes, and approximately one hundred candidates for p53-binding sequences, are present in human chromosomes. It is thought the bioactivity of this vast number of p53 target genes is reflected in p53's tumor suppressing actions resulting from by p53-mediated cell cycle arrest, DNA repair and apoptosis. Therefore, identifying additional p53 target genes is important in elucidating the mechanisms of tumorigenesis, and cell protection from genotoxic stresses.
Induction of apoptosis is known to be the most important of p53's tumor suppressing functions, and this activity is being used to kill cancer cells in human patients (Levine, Cell: 88, 323-331, 1997). However, despite this kind of evidence, the mechanism of p53-mediated apoptosis has yet to be elucidated.
BAX, Fas, Killer/DR5, and PIGs are recognized as candidates for mediating p53-dependent apoptosis, and their functions are currently being investigated. However, acting alone, any one of these candidates is insufficient to induce apoptosis. The current inventors have found p53AIP1, a novel target gene for p53, which differs from the candidates described above in that it can induce apoptosis independently when over-expressed in some cancer cells. Blocking p53AIP1 expression inhibits p53-mediated apoptosis (Oda, K., Arakawa, H., Tanaka, T., Matsuda, K., Tanikawa, C., Mori, T., Nishimori, H., Tamai, K., Tokino, T., Nakamura, Y., and Taya, Y. (2000) p53AIP1, a potential mediator of p53-dependent apoptosis, and its regulation by Ser-46-phosphorylated p53. Cell 102, 849-862) These results suggested that p53AIP1 was a crucial mediator in the mechanism of p53-dependent apoptosis.
Growing evidence suggests that the biological activity of p53 is determined by modifications such as phosphorylation, acetylation, and the like (Giaccia, A. J. and Kastan, M. B. (1998). The complexity of p53 modulation: emerging patterns from divergent signals. Genes Dev. 19, 2973-2983). For example, there are reports indicating that phosphorylation of the p53 protein at its Ser15 and Ser20 residues is important in p53 activation in response to DNA damage (Shieh, S. Y., Ikeda, M., Taya, Y., and Prives, C. (1997) DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2. Cell 91, 325-334; Shieh, S. Y., Taya, Y., and Prives, C. (1999) DNA damage-inducible phosphorylation of p53 at N-terminal sites including a novel site, Ser20, requires tetramerization. EMBO J. 18, 1815-1823). There are also reports that acetylation of the p53 C-terminal domain enhances DNA binding activity (Gu, W. and Roeder, R. G. (1997). Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain. Cell 90, 595-606). In addition, ATM and CHK2 proteins have been proposed as strong candidates for a kinase involved in the phosphorylation of p53 at Ser15 and Ser20 respectively (Sarkaria, J. N., Busby, E. C., Tibbetts, R. S., Roos, P., Taya, Y., Karnitz, L. M, and Abraham, R. T. (1999). Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine. Cancer Res. 59, 4375-4382; Hirao, A., Kong, Y. Y., Matsuoka, S., Wakeham, A., Ruland, J., Yoshida, H., Liu, D., Elledge, S. J., and Mak, T. W. (2000). DNA damage-induced activation of p53 by the checkpoint kinase Chk2. Science 287, 1824-1827). Furthermore, it has been shown that phosphorylation of the Ser46 residue is essential for p53-induced apoptosis (Oda et al., supra).
Based on the various findings described above, the present inventors speculated that p53 determines whether cells with damaged DNA will survive or be killed, such that cells which are extremely damaged or have been exposed to danger are eliminated through phosphorylation of p53 at Ser46, and induction of p53AIP1.
Because known p53 targets do not sufficiently explain this speculation, the present inventors used a method capable of directly cloning human chromosome-derived p53-binding sequences for further screening of p53 targets. The present inventors identified four novel target genes and a GPI-anchored molecule-like protein (GML), whose expressions are induced by wild-type p53 (Furuhata, T., Tokino, T., Urano, T., and Nakamura, Y. (1996). Isolation of a novel GPI-anchored gene specifically regulated by p53; correlation between its expression and anti-cancer drug sensitivity. Oncogene 13, 1965-1970; Kimura, Y., Furuhata, T., Urano, T., Hirata, K., Nakamura, Y., and Tokino, T. (1997). Genoa structure and chromosomal localization of GML (GPI-anchored molecule-like protein), a gene induced by p53. Genomics 41, 477-480), P2XM (Urano, T., Nishimori,. H., Han, H., Furuhata, T., Kimura, Y., Nakamura, Y., and Tokino, T. (1997). Cloning of P2XM, a novel human P2X receptor gene regulated by p53. Cancer Res. 57, 3281-3287), BAI1 (Nishimori, H., Shiratsuchi, T., Urano, T., Kimura, Y., Kiyono, K., Tatsumi, K., Yoshida, S., Ono, M., Kuwano, M., Nakamura, Y., and Tokino, T. (1997). A novel brain-specific p53-target gene, BAI1, containing thrombospondin type 1 repeats inhibits experimental angiogenesis. Oncogene 15, 2145-2150) and CSR (Han, H. J., Tokino, T., and Nakamura, Y. (1998). CSR, a scavenger receptor-like protein with a protective role against cellular damage caused by UV irradiation and oxidative stress. Hum. Mol. Genet. 7, 1039-1046).
The present inventors isolated a p53-inducible transcript by establishing a cell line in which p53 expression is regulated under set conditions, and then applied differential display techniques to that cell line (Takei, Y., Ishikawa, S., Tokino, T., Muto, T., and Nakamura, Y. (1998). Isolation of a novel TP53 target gene from a colon cancer cell line carrying a highly-regulated wild-type TP53 expression system. Genes Chromosomes Cancer 23, 1-9) Using this approach, three additional novel p53 target genes were identified: TP53TG1 (Takei et al., supra), TP53TG3 (Ng, C. C., Koyama, K., Okamura, S., Kondoh, H., Takei, Y., and Nakamura, Y. (1999). Isolation and characterization of a novel TP53-inducible gene, TP53TG3. Genes Chromosomes Cancer 26, 329-335) and p53R2 (Tanaka, H., Arakawa, H., Yamaguchi, T., Shiraishi, K., Fukuda, S., Matsui, K., Takei, Y., and Nakamura, Y. (2000). A ribonucleotide reductase gene involved in p53-dependent cell-cycle checkpoint for DNA damage. Nature 404, 42-49). Genes already known to be activated or suppressed by p53 were also identified during this approach.